Effect of equilibration period on the viability of frozen‐thawed mouse morulae after rapid freezing

Abstract

Mouse morulae were frozen with 1.5-4.0 M glycerol + 0.25 M lactose solution by direct plunging into liquid nitrogen vapor 0.5-30 min after equilibration at room temperature. After thawing, embryos were cultured in vitro, and the highest survival rates were obtained after exposure for 3 min at 3.0 and 4.0 M and for 5 min at 1.5 and 2.0 M glycerol levels. Significant reductions in the survival rates (P less than 0.05) were observed when equilibration periods were extended for 3-5 min at 3.0 and 4.0 M and for 5-10 min at 1.5 and 2.0 M glycerol levels. These results clearly demonstrate that the equilibration time of embryos in glycerol-lactose mixture is one of the most important factors in the present rapid freezing conditions. To clarify the factors that lower embryo viability after prolonged equilibration, we performed further experiments on the effects of exposure to glycerol-lactose mixture on the developmental potential of embryos without freezing and on the volume changes of embryos during the exposure to glycerol solution with or without lactose. It was suggested that the detrimental effects of prolonged equilibration are due not only to the toxicity and osmotic injury of higher concentrations of cryoprotectant solution but also to the influx of water into embryonic cells caused by the hypotonic salt concentration of the extracellular (freezing) solution.

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“…Similar results, that the mouse morulae exhibited high survival rates after rapid freezing have been reported by a limited number of authors (16,19,20). However, morula/compact embryos were never classified into substages as this article does.…”

Birth of piglets after OPS vitrification and transfer of compacted morula stage embryos with intact zona pellucida

“…In rabbit, vitrified morulae were transferred and the survival rate (foetus or kits) observed in four studies were 41%, 24%, 65% and 31% respectively [8,9,10,20]. In mice, only in vitro development after vitrification was tested [4,16] and a high development rate was obtained up to 86% and 82%, respectively. In pigs, like in mice, in vitro development was studied [6,7,12] but few morulae developed into blastocysts, 6%, 33%, 0% respectively.…”

Bovine Oocytes and Early Embryos Express mRNA Encoding Glycerol Kinase but Addition of Glycerol to the Culture Media Interferes with Oocyte Maturation

“…Although the underlying mechanism of the interference is not clear, the presence of glycerol in culture media may be toxic to the oocyte. It has been reported that exposure to cryopreservation medium containing more than 1.0 M glycerol decreases viability of mouse embryos [22]. In the present study, glycerol was added to a final concentration of 1 mM to test whether it could be used as an energy substrate by the oocyte and embryo.…”